Method for detecting glucagon-like peptide-1 antagonists and agonists

ABSTRACT

The present invention relates to a recombinant glucagon-like peptide-1 (GLP-1) receptor, to a DNA construct which comprises a DNA sequence encoding a GLP-1 receptor, to methods of screening for antagonists of GLP-1 activity, and to the use of the GLP-1 receptor for screening for antagonists of GLP-1 activity.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part of U.S. Ser. No. 08/142,439filed Nov. 24, 1993, now U.S. Pat. No. 5,670,360 which is a nationalapplication of PCT/EP93/00697 filed Mar. 23, 1993, and claims priorityunder 35 U.S.C. 119 of Danish Application serial no. 398/92 filed Mar.25, 1992, the contents of which are fully incorporated herein byreference.

FIELD OF THE INVENTION

The present invention relates to a recombinant glucagon-like peptide-1(GLP-1) receptor, to a DNA construct which comprises a DNA sequenceencoding a GLP-1 receptor, to methods of screening for antagonists ofGLP-1 activity and to methods for functional screening for agonists ofGLP-1 activity.

BACKGROUND OF THE INVENTION

As used in the present specification the designation GLP-1 comprisesGLP-1(7-37) as well as GLP-1(7-36)amide.

Glucose-induced insulin secretion is modulated by a number of hormonesand neurotransmitters. In particular, two gut hormones, glucagon-likepeptide-1 (GLP-1) and gastric inhibitory peptide (GIP) potentiate theeffect of glucose on insulin secretion and are thus calledgluco-incretins (Dupre, in The Endocrine Pancreas, E. Samois Ed. (RavenPress, New York, (1991), 253-281) and Ebert and Creutzfeld, (DiabetesMetab. Rev. 3, (1987)). Glucagon-like peptide-1 is a gluco-incretin bothin rat and in man (Dupre and Ebert and Creutzfeld, vide supra, andKreymann et al. (Lancet 2 (1987), 1300)). It is part of thepreproglucagon molecule (Bell et al. Nature 304 (1983), 368) which isproteolytically processed in intestinal L cells to GLP-1(1-37) andGLP-1(7-36)amide or GLP-1(7-37) (Mojsov et al. (J.Biol.Chem. 261 (1986),11880) and Habener et al.: The Endocrine Pancreas E. Samois Ed. (RavenPress, New York (1991), 53-71). Only the truncated forms of GLP-1 arebiologically active and both have identical effects on insulin secretionin beta cells (Mojsov et al. J.Clin.Invest 79 (1987), 616) and Weir etal. (Diabetes 38 (1989), 338). They are the most potent gluco-incretinsso far described and are active at concentrations as low as one to tenpicomolar. The stimulatory effect of these gluco-incretin hormonesrequires the presence of glucose at or above the normal physiologicalconcentration of about 5 mM and is mediated by activation of adenylatecyclase and a rise in the intracellular concentration of cyclic AMP(Drucker et al. Proc.Natl.Acad.Sci. USA 84 (1987), 3434) and G'ke et al.(Am.J.Physiol. 257 (1989), G397). GLP-1 has also a stimulatory effect oninsulin gene transcription (Drucker et al. Proc.Natl.Acad.Sci. USA 84(1987), 3434). In a rat model of non-insulin-dependent diabetes mellitus(NIDDM) is associated with a reduced stimulatory effect of GLP-1 onglucose-induced insulin secretion (Suzuki et al. Diabetes 39 (1990),1320). In man, in one study, GLP-1 levels were elevated in NIDDMpatients both in the basal state and after glucose ingestion; however,following a glucose load there was only a very small rise in plasmainsulin concentration (qrskov et al. J.Clin.Invest. 87 (1991), 415). Arecent study (Nathan et al. Diabetes Care 15 (1992), 270) showed thatGLP-1 infusion could ameliorate postprandial insulin secretion andglucose disposal in NIDDM patients. Thus, as a further step inunderstanding the complex modulation of insulin secretion by guthormones and its dysfunction in diabetes, we isolated and characterizeda complementary DNA for the beta cell GLP-1 receptor and showed that itis part of a new family of G-coupled receptors.

DESCRIPTION OF THE INVENTION

The present invention relates to a recombinant glucagon-like peptide-1(GLP-1) receptor.

More preferably, the invention relates to a GLP-1 receptor whichcomprises the amino acid sequence shown in SEQ ID No. 1, or an analoguethereof binding GLP-1 with an affinity constant, K_(D), below 100 nM,preferably below 10 nM. In the present context, the term "analogue" isintended to indicate a naturally occurring variant (including oneexpressed in other animal species, in particular human) of the receptoror a "derivative" i.e. a polypeptide which is derived from the nativeGLP-1 receptor by suitably modifying the DNA sequence coding for thevariant, resulting in the addition of one or more amino acids at eitheror both the C- and N-terminal ends of the native amino acid sequence,substitution of one or more amino acids at one or more sites in thenative amino acid sequence, deletion of one or more amino acids ateither or both ends of the native sequence or at one or more siteswithin the native sequence, or insertion of one or more amino acids inthe native sequence.

In another aspect, the present invention relates to a DNA constructwhich comprises a DNA sequence encoding the GLP-1 receptor of theinvention, as well as a recombinant expression vector carrying the DNAconstruct and a cell containing said recombinant expression vector.

In one embodiment of the invention, the GLP-1 receptor molecule may beprovided in solubilised and/or reconstituted form.

In the present context "solubilised" is intended to indicate a receptoras present in detergent-solubilised membrane preparations."Reconstituted" is intended to indicate a receptor solubilised in theprescience of essential cofactors, e.g. G-protein. In this embodimentthe receptor may be in a reconstituted micellar form.

The DNA construct of the invention encoding the GLP-1 receptorpreferably comprises the DNA sequence shown in SEQ ID No. 1, or at leasta DNA sequence coding for a functional analogue thereof binding GLP-1with an affinity below 100 nM, preferably below 10 nM or a suitablemodification thereof. Examples of suitable modifications of the DNAsequence are nucleotide substitutions which do not give rise to anotheramino acid sequence of the GLP-1 receptor, but which may correspond tothe codon usage of the host organism into which the DNA construct isintroduced or nucleotide substitutions which do give rise to a differentamino acid sequence and therefore, possibly, a different proteinstructure without, however, impairing the properties of the nativevariant. Other examples of possible modifications are insertion of oneor several nucleotides into the sequence, addition of one or severalnucleotides at either end of the sequence, or deletion of one or severalnucleotides at either end or within the sequence.

Another example of a DNA construct of the invention is one which encodesa GLP-1 receptor variant particularly suitable for solubilisation andreconstitution.

The DNA construct of the invention encoding the present GLP-1 receptormay be prepared synthetically by established standard methods, e.g. thephosphoamidite method described by Beaucage and Caruthers, TetrahedronLetters 22 (1981), 1859-1869, or the method described by Matthes et al.,EMBO Journal 3 (1984), 801-805. According to the phosphoamidite method,oligonucleotides are synthesized, e.g. in an automatic DNA synthesizer,purified, annealed, ligated and cloned in suitable vectors.

The DNA construct of the invention may also be of genomic or cDNAorigin, for instance obtained by preparing a genomic or cDNA library andscreening for DNA sequences coding for all or part of the GLP-1 receptorof the invention by hybridization using synthetic oligonucleotide probesin accordance with standard techniques (cf. Sambrook et al., MolecularCloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor, 1989). Inthis case, a genomic or cDNA sequence encoding the GLP-1 receptor may bemodified at a site corresponding to the site(s) at which it is desiredto introduce amino acid substitutions, e.g. by site-directed mutagenesisusing synthetic oligonucleotides encoding the desired amino acidsequence for homologous recombination in accordance with well-knownprocedures.

Finally, the DNA construct may be of mixed synthetic and genomic, mixedsynthetic and cDNA or mixed genomic and cDNA origin prepared by ligatingfragments of synthetic, genomic or cDNA origin (as appropriate), thefragments corresponding to various parts of the entire DNA construct, inaccordance with standard techniques. The DNA construct may also beprepared by polymerase chain reaction using specific primers, forinstance as described in U.S. Pat. No. 4,683,202 or Saiki et al.,Science 239 (1988), 487-491.

The recombinant expression vector into which the DNA construct of theinvention is inserted may be any vector which may conveniently besubjected to recombinant DNA procedures, and the choice of vector willoften depend on the host cell into which it is to be introduced. Thus,the vector may be an autonomously replicating vector, i.e. a vectorwhich exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g. a plasmid. Alternatively,the vector may be one which, when introduced into a host cell, isintegrated into the host cell genome and replicated together with thechromosome(s) into which it has been integrated.

In the vector, the DNA sequence encoding the GLP-1 receptor of theinvention should be operably connected to a suitable promoter sequence.The promoter may be any DNA sequence which shows transcriptionalactivity in the host cell of choice and may be derived from genesencoding proteins either homologous or heterologous to the host cell.Examples of suitable promoters for directing the transcription of theDNA encoding the GLP-1 receptor of the invention in mammalian cells arethe SV40 promoter (Subramani et al., Mol. Cell Biol. 1 (1981), 854-864),the MT-1 (metallothionein gene) promoter (Palmiter et al., Science 222(1983), 809-814) or the adenovirus 2 major late promoter. A suitablepromoter for use in insect cells is the polyhedrin promoter (Vasuvedanet al., FEBS Lett. 311, (1992) 7-11). Suitable promoters for use inyeast host cells include promoters from yeast glycolytic genes (Hitzemanet al., J. Biol. Chem. 255 (1980), 12073-12080; Alber and Kawasaki, J.Mol. Appl. Gen. 1 (1982), 419-434) or alcohol dehydrogenase genes (Younget al., in Genetic Engineering of Microorganisms for Chemicals(Hollaender et al, eds.), Plenum Press, New York, 1982), or the TPI1(U.S. Pat. No. 4,599,311) or ADH2-4c (Russell et al., Nature 304 (1983),652-654) promoters. Suitable promoters for use in filamentous fungushost cells are, for instance, the ADH3 promoter (McKnight et al., TheEMBO J. 4 (1985), 2093-2099) or the tpiA promoter.

The DNA sequence encoding the GLP-1 receptor of the invention may alsobe operably connected to a suitable terminator, such as the human growthhormone terminator (Palmiter et al., op. cit.) or (for fungal hosts) theTPI1 (Alber and Kawasaki, op. cit.) or ADH3 (McKnight et al., op. cit.)terminators. The vector may further comprise elements such aspolyadenylation signals (e.g. from SV40 or the adenovirus 5 Elb region),transcriptional enhancer sequences (e.g. the SV40 enhancer) andtranslational enhancer sequences (e.g. the ones encoding adenovirus VARNAs).

The recombinant expression vector of the invention may further comprisea DNA sequence enabling the vector to replicate in the host cell inquestion. An example of such a sequence (when the host cell is amammalian cell) is the SV40 origin of replication. The vector may alsocomprise a selectable marker, e.g. a gene the product of whichcomplements a defect in the host cell, such as the gene coding fordihydrofolate reductase (DHFR) or one which confers resistance to adrug, e.g. neomycin, hygromycin or methotrexate.

The procedures used to ligate the DNA sequences coding for the GLP-1receptor of the invention, the promoter and the terminator,respectively, and to insert them into suitable vectors containing theinformation necessary for replication, are well known to persons skilledin the art (cf., for instance, Sambrook et al., op.cit.).

The host cell into which the expression vector of the invention isintroduced may be any cell which is capable of producing the GLP-1receptor of the invention and is preferably a eukaryotic cell, such asinvertebrate (insect) cells or vertebrate cells, e.s. Xenopus laevisoocytes or mammalian cells, in particular insect and mammalian cells.Examples of suitable mammalian cell lines are the COS (ATCC CRL 1650),BHK (ATCC CRL 1632, ATCC CCL 10), CHL (ATCC CCL39) or CHO (ATCC CCL 61)cell lines. Methods of transfecting mammalian cells and expressing DNAsequences introduced in the cells are described in e.g. Kaufman andSharp, J. Mol. Biol. 159 (1982), 601-621; Southern and Berg, J. Mol.Appl. Genet. 1 (1982), 327-341; Loyter et al., Proc.Natl.Acad.Sci. USA79 (1982), 422-426; Wigler et al., Cell 14 (1978), 725; Corsaro andPearson, Somatic Cell Genetics 7 (1981), 603, Graham and van der Eb,Virology 52 (1973), 456; and Neumann et al., EMBO J. 1 (1982), 841-845.

Alternatively, fungal cells (including yeast cells) may be used as hostcells of the invention. Examples of suitable yeasts cells include cellsof Saccharomyces spp. or Schizosaccharomyces spp., in particular strainsof Saccharomyces cerevisiae. Examples of other fungal cells are cells offilamentous fungi, e.g. Aspergillus spp. or Neurospora spp., inparticular strains of Aspergillus oryzae or Aspergillus niger. The useof Aspergillus spp. for the expression of proteins is described in,e.g., EP 272 277.

The GLP-1 receptor according to the invention may be produced by amethod which comprises culturing a cell as described above in a suitablenutrient medium under conditions which are conducive to the expressionof the GLP-1 receptor, and recovering the GLP-1 receptor from theculture. The medium used to culture the cells may be any conventionalmedium suitable for growing mammalian cells, such as a serum-containingor serum-free medium containing appropriate supplements. Suitable mediaare available from commercial suppliers or may be prepared according topublished recipes (e.g. in catalogues of the American Type CultureCollection).

If the GLP-1 receptor has retained the transmembrane and (possibly) thecytoplasmic region of the native variant, it will be anchored in themembrane of the host cell, and the cells carrying the GLP-1 receptor maybe used as such in the screening or diagnostic assay. Alternatively, thereceptor may be a component of membrane preparations, e.g. insolubilised and/or reconstituted form as defined above.

The present invention also provides methods for detecting GLP-1antagonists/inverse agonists. Within the context of the invention, anGLP-1 antagonist/inverse agonist is understood to refer to a moleculethat reduces the GLP-1 stimulated response within a given cell.

Within one aspect of the present invention, methods are provided fordetecting the presence of GLP-1 antagonists comprising the steps of (a)exposing the compound in the presence of a GLP-1 agonist to arecombinant GLP-1 receptor coupled to a response pathway underconditions and for a time sufficient to allow binding of the compound tothe receptor and an associated response through the pathway, and (b)detecting a reduction in the stimulation of the response pathwayresulting from the binding of the compound to the GLP-1 receptor,relative to the stimulation of the response pathway by the GLP-1 agonistalone and therefrom determining the presence of a GLP-1antagonist/inverse agonist. Within the context of the present invention,GLP-1 agonists include molecules (including GLP-1 itself) capable ofbinding to a GLP-1 receptor, and which stimulate a response pathwaywithin a cell.

A variety of compounds may be screened utilizing such methods.Representative examples include blocking antibodies, GLP-1 peptides andGLP-1 analogs (including both peptides and non-peptides).

The compounds are exposed to a recombinant GLP-1 receptor in thepresence of a GLP-1 agonist under conditions and for a time sufficientto allow binding of the compound to the receptor, and an associatedresponse though the pathway. As utilized in the present invention,conditions and times sufficient for the binding of theantagonist/inverse agonist to the receptor will vary with the source ofthe receptor, however, conditions suitable for the binding generallyoccur between 4° and 40° C. in a buffer solution and within a pH rangeof 5 and 9, preferable between 6.8 and 8. Sufficient time for thebinding and response will generally be between 5 and 200 minutes afterexposure.

Once the compound has been exposed to a recombinant GLP-1 receptor inthe presence of a GLP-1 agonist, under conditions and for a timesufficient to allow binding of the compound to the receptor, a reductionin the stimulation of the response pathway may be detected if thecompounds competes with the GLP-1 agonist for the recombinant GLP-1receptor. Within one embodiment of the invention, the response pathwayis a membrane bound adenylate cyclase response pathway, and the step ofdetecting comprises measuring a reduction in cAMP production by themembrane bound adenylate cyclase response pathway, relative to the cAMPproduction in the presence of GLP-1 agonist alone. Adenylate cyclaseactivity assays may be carried out, for example, utilizing method(s)described by Knudsen et al. (Eur. J. Pharmacol., 318, 429-435(1996)).Alternatively, cAMP may be measured by any other assay. Generally, theseassays are well known in the art.

In another embodiment of the invention, the present invention relates toa method for functional screening for the presence of GLP-1 agonists,the method comprising the steps of:

(a) exposing a compound to a recombinant GLP-1 receptor coupled to aresponse pathway under conditions and for a time sufficient to allowbinding of the compound to the receptor and an associated responsethrough the pathway; and

(b) detecting stimulation of the response pathway relative to the nonstimulated pathway.

With a preferred embodiment of the invention a DNA construct is providedcomprising a cAMP response element such as proenkephalin cAMP responseelement, which is operately linked to a luciferase cDNA. The DNAconstruct comprising the luciferase cDNA is then stably transfected intoa host cell. The host cell is then transfected with a second DNAconstruct containing a first DNA segment encoding the GLP-1 receptoroperably linked to additional DNA segments necessary for the expressionof the receptor. Upon binding of the GLP-1 receptor agonist, theelevated cAMP levels induce the expression of luciferase. The luciferaseis exposed to luciferin, and the photons released during the oxidationof luciferin by the luciferase is measured.

The solid support employed in the screening methods of the inventionpreferably comprises a polymer. The support may in itself be composed ofthe polymer or may be composed of a matrix coated with the polymer. Thematrix may be of any suitable material such as glass, paper or plastic.The polymer may be selected from the group consisting of a plastic (e.g.latex, a polystyrene, polyvinylchloride, polyurethane, polyacrylamide,polyvinylalcohol, nylon, polyvinylacetate, and any suitable copolymerthereof), cellulose (e.g. various types of paper, such as nitrocellulosepaper and the like), a silicon polymer (e.g. siloxane), a polysaccharide(e.g. agarose or dextran), an ion exchange resin (e.g. conventionalanion or cation exchange resins), a polypeptide such as polylysine, or aceramic material such as glass (e.g. controlled pore glass).

The physical shape of the solid support is not critical, although someshapes may be more convenient than others for the present purpose. Thus,the solid support may be in the shape of a plate, e.g. a thin layer ormicrotiter plate, or a film, strip, membrane (e.g. a nylon membrane or acellulose filter) or solid particles (e.g. latex beads or dextran oragarose beads). In a preferred embodiment, the solid support is in theform of wheat germ agglutinin-coated SPA beads (cf. U.S. Pat. No.4,568,649).

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention is further illustrated in the following exampleswith reference to the appended drawings in which

FIG. 1A and FIG. 1B which is a continuation of FIG. 1A together show theamino acid sequence of the rat GLP-1 receptor (SEQ ID NO:1) in acomparison with the sequence of the rat secreting receptor (SECR) (SEQID NO:5), the opossium parathyroid hormone receptor (PTHR) (SEQ ID NO:6)and the porcine calcitonin receptor (CTR1) (SEQ ID NO:7). The GLP-1receptor has three N glycosylation sites in the extracellular domain(arrows). Four cysteines are conserved at identical places in the fourreceptor (boxes). Note the otherwise very divergent sequences in thispart of the molecules as well as in the COOH-terminal cytoplasmic tail.Sequence identities are denoted by stars and homologies by dots. Thelocation of the putative transmembrane domains are indicated byhorizontal bars above the sequences.

FIG. 2 shows binding of ¹²⁵ I-GLP-1 to COS cells transfected with thepGLPR-16 plasmid. Specific binding reaches saturation at 1 to 10 nMGLP-1. Insert: Scatchard analysis of GLP-1 binding.

FIG. 3 shows binding of ¹²⁵ I-GLP-1 to INS-1 cells. Specific bindingreaches saturation at 1 to 10 nM GLP-1. Insert: Scatchard analysis ofGLP-1 binding.

Fitting of the curves in FIGS. 2 and 3 were performed with the LIGANDprogram (McPherson, Kinetic, EBDA, Ligand, Lowry. A Collection ofradioligand analysis programs (Elsevier, Amsterdam, 1985)).

FIG. 4 shows displacement of ¹²⁵ I-GLP-1 binding to COS cellstransfected with the rat GLP-1 receptor cDNA. Transfected cells wereincubated with 20 pM ¹²⁵ I-GLP-1 in the presence of increasingconcentrations of cold peptides. Each point was measured in duplicateand the experiments repeated three times for GLP-1, GIP and glucagon andonce for VIP and secreting.

FIG. 5 shows stimulation of cyclic AMP formation in COS cellstransfected with the rat GLP-1 receptor cDNA. COS cells were transfectedwith the pcDNA-1 vector alone (open bars) or the pGLPR-1 plasmid(stripped bar) and incubated in the absence or the presence of GLP-1 atthe indicated concentration. cAMP production was measured in triplicatewith a radioimmunoassay (Amersham).

FIG. 6 shows tissue specificity of GLP-1 receptor expression assessed byNorthern blotting of RNA from different tissues and from the INS-1 cellline. Ten micrograms of total RNA was analyzed on each lane. Two majorRNA species of 2.7 and 3.6 kb were detected in all tissues in which thereceptor was detected. The position of the migration of the ribosomalRNAs is indicated to the left of the picture.

FIG. 7 is a comparison of rat GLP-1 receptor (SEQ ID No.1) amino acidsequence (rat) and a partial amino acid sequence of the human GLP-1receptor (SEQ ID No. 3) (human).

The present invention is further illustrated in the following exampleswhich is not intended to be in any way limiting to the scope of theinvention as claimed.

EXAMPLE 1 Molecular Cloning and Characterisation of the Rat Islet GLP-1Receptor cDNA.

A rat pancreatic islet cDNA library was constructed in the pcDNA-1expression vector (Rat pancreatic islets were prepared according toGotoh et al. (Transplantation 43 (1985), 725). PolyA+ RNA was preparedand the cDNA library was constructed in the pcDNA-1 vector (In Vitrogen)as described by Aruffo and Seed (Proc.Natl.Acad.Sci. USA 84 (1987),8573) and Lin et al. (Proc.Natl.Acad.Sci. USA 88 (1991), 3185). PlasmidDNA was prepared from pools of five to eight thousands bacterial clones(Maniatis et al., Molecular Cloning. A Laboratory Manual. Cold SpringHarbor Laboratory, 1982) and transfected into COS cells (Sompayrac andDana, Proc.Natl.Acad.Sci. USA 78 (1981), 7575). The presence of GLP-1receptor expressed in COS cells was assessed by binding of theradioiodinated peptide followed by photographic emulsion autoradiographyand screening by dark field microscopy (Gearing et al. EMBO J. 8 (1989),3667). GLP-1(7-36)amide, as well as the other peptides, were purchasedfrom Peninsula Laboratories. Iodination was performed by the iodinemonochloride method (Contreras et al. Meth.Enzymol. 92 (1983), 277), thepeptide was purified by passage over Sephadex G-10 followed byCM-Sepharose and specific activity was determined by the selfdisplacement technique (Calvo et al. Biochem. 212 (1983), 259). A 1.6 kbcDNA clone (pGLPR-1) was isolated by subfractionation of an originalpositive pool and was used to isolate, by DNA hybridization screening,two additional clones from primary positive pools. These plasmids(pGLPR-16 and -87) had inserts of 3.0 and 2.0 kb, respectively.Transfection of these clones into COS cells generated high affinity(K_(D) =0.6 nM) binding sites for GLP-1 (FIG. 2). This affinity iscomparable to that seen for binding of GLP-1 to the rat insulinoma cellline INS-1 (Asfari et al. Endocrinology 130 (1992), 167) (K_(D) =0.12nM; FIG. 3). In both cases a single high affinity binding component wasdetected. The binding to GLP-1 receptor transfected COS cells reached aplateau between 1 and 10 nM. At concentrations above 10 nM a second,high capacity, low affinity, binding component was detected. Althoughspecifically displacable by cold GLP-1, this binding was also present inCOS cells transfected with the expression vector alone and was thereforenot further characterized.

Binding of GLP-1 to the receptor expressed in COS cells was displaced bycold GLP-1 with a 50 percent displacement achieved at 0.5 to 1 nM (FIG.4). Other peptide hormones of related structure such as secreting,gastric inhibitory peptide (GIP) and vasoactive intestinal peptide (VIP)(Dupre in The Endocrine Pancreas, E. Samois Ed. (Raven Press, New York,(1991), 253-281) and Ebert and Creutzfeld, Diabetes Metab. Rev. 3,(1987) did not displace binding. Glucagon could displace the binding by50 percent but only at a concentration of one micromolar (FIG. 4). Theaddition of subnanomolar concentrations of GLP-1 to transfected COScells stimulated the production of cyclic AMP indicating that thereceptor was functionally coupled to activation of adenylate cyclase(FIG. 5).

DNA sequence analysis of the rat GLP-1 receptor cDNA revealed a majoropen reading frame coding for a 463 amino acid polypeptide (SEQ ID No.1). Hydrophaphy plot analysis indicated the presence of anamino-terminal hydrophobic region most probably representing a leadersequence. This hydrophobic segment is followed by a hydrophilic domainof about 120 amino acids which contains three N-linked glycosylationsites. Seven hydrophobic segments are present which may formtransmembrane domains. Search for sequence identities showed the GLP-1receptor to be homologous to the secreting receptor (Ishihara et al.EMBO J. 10 (1991), 1635) (40 percent identity), the parathyroid hormonereceptor (Jhppner et al. (Science 254 (1991), 1024) (32.4 percentidentity) and the calcitonin receptor (Lin et al. Science 254 (1991),1022) (27.5 percent identity) (FIG. 1). These four receptors do notshare any significant sequence homology with other known members of theG-coupled receptor family and are characterized by a relatively longamino terminal, probably extracellular, domain. The sequence of theextracellular domain is unique for each receptor, yet four cysteines areperfectly conserved (boxes in FIG. 1). A fifth cysteine at position 126of the GLP-1 receptor is also conserved in the parathyroid andcalcitonin receptors and at a similar location in the secreting receptor(position 123). The highest sequence identity between the four proteinsresides in the transmembrane domains. The carboxyl terminal,cytoplasmic, ends of each receptor are also very different. Thesereceptors all stimulate the production of cyclic AMP in response toligand binding (Ishihara et al. EMBO J. 10 (1991), 1635), Jhppner et al.(Science 254 (1991), 1024) and Lin et al. Science 254 (1991), 1022) andare presumably coupled to the cyclase via Gs". In that respect, it isinteresting to note that a sequence motif present in the thirdcytoplasmic loop of the GLP-1 receptors (RLAK, present just before thesixth transmembrane domain) is very similar to a motif of the beta2adrenergic receptor (KALK) present at the same location and whose basicamino acids have been shown to be important in the coupling of thereceptor to Gs" (Okamoto et al. Cell 67 (1991), 723). Moreover, in thebeta2 adrenergic receptor, this motif is preceeded by a basic amino acidlocated twelve amino acid toward the amino-terminal end. This basicamino acid is also required at this particular distance for efficientcoupling to Gs". In the GLP-1 receptor a lysine residue is also presentat a similar location. This suggests that, despite the very low overallsequence identity, a structural feature may have been conserved in thethird cytoplasmic loop between the two receptors which, may be requiredfor the coupling of receptor to the Gs" protein.

Determination of the tissue distribution of the GLP-1 receptor wasperformed by Northern blot analysis. Northern blot analysis wasperformed with 10:g of total RNA (Chomczynski and Sacchi, Anal.Biochem.126 (1987), 156) denatured with glyoxal (McMaster and Carmichael,Proc.Natl.Acad.Sci. USA 74 (1977), 4835) separated on a 1% agarose geland transferred to Nylon membranes (Thomas, Proc.Natl.Acad.Sci. USA 77(1980), 5201). Hybridization was performed with the random primedlabelled (Feinberg and Vogelstein, Anal.Biochem. 132 (1983), 6) 1,6 kbpGLPR-1 insert. Two mRNAs of 2.7 and 3.6 kb could be detected inpancreatic islets as well as in rat insulinoma cell lines (INS-1), instomach and in lung (FIG. 6). No GLP-1 receptor mRNA could be detectedin brain, liver, thymus, muscle, intestine and colon. The presence ofthe GLP-1 receptor has been reported in stomach where the peptideinhibits acid secretion by parietal cells in in vivo experiments(Schjoldager et al. Dig.Dis.Sci. 34 (1989), 703) but stimulates acidsecretion on isolated parietal glands (Schmidtler et al. Am.J.Physiol.260 (1991), G940). Binding sites for GLP-1 have also ben reported inlung membrane preparations (Richter et al. FEBS Letter 1 (1990), 78) butthe role of the hormone on lung physiology is not known.

A stable cell line expressing the cloned rat GLP-1 receptor wasestablished by Ca-phosphate mediated transfection (Maniatis et al.,Molecular Cloning. A Laboratory Manual. Cold Spring Harbour Laboratory,1989) of the CHL cell line (ATCC CCL39). The plasmid, pGLPR-1, whichcontains a 1.6 kb rat GLP-1 receptor cDNA insert cloned in the pcDNA-1vector, was cotransfected with the pWL-neo plasmid (Stratagene, LaJolla, Calif.) into CHL cells. The pWL-neo plasmid contains the neomycinresistance gene. Stable clones were selected in medium containing 0.8mg/ml G418. A stable transformant expressing an estimate of 70.000 ratGLP-1 receptors pr cell was selected by this scheme and furtherpropagated in the presence of 80:M G418. Membranes from thistransformant was subsequently used in thehigh-volume-throughput-screening (HVTS) assay as described in Example 3.Characterization of the receptor expressed by the GLP-1 R/CHL cell lineled to an estimated Kd of 0.8 nM for whole cells, 2.3 nM for cellmembranes using ¹²⁵ I-GLP-1(7-36)amide as radioligand.

EXAMPLE 2 Molecular cloning of the human islet GLP-1 receptor cDNA.

Human islets were prepared as described (Ricordi et al., Diabetes 37(1988), 413-420), and poly-A⁺ RNA was isolated by affinitychromatography by published methods (Gonda et al., Mol. Cell. Biol. 2(1982) 617-624).

A human islet cDNA library was constructed in the 8ZAPII vector fromStratagene (La Jolla, Calif.). Briefly, double stranded cDNA wassynthesized as previously described (Aruffo and Seed, 84 (1987),8573-8577; Thorens, Proc.Natl.Acad.Sci., USA 89 (1992), 8641-8645), andEcoRI/NotI adaptors (Stratagene, La Jolla, Calif.) were added with T₄DNA ligase.

The resulting cDNA molecules were phosphorylated with T₄ polynucleotidekinase before size fractionation on potassium acetate gradients (Aruffoand Seed, 84 (1987), 8573-8577).

Double stranded cDNA with a size above 1.6 kb was ligated into 8ZAPIIarms (Stratagene, La Jolla, Calif.), packaged inr8 phage and grown on alawn of XL-1 Blue E. coli cells as described in protocols fromStratagene.

The cDNA library was screened by hybridization to a ³² P labelled DNAfragment from the rat GLP-1 receptor cDNA by previously describedmethods (Maniatis et al., Molecular Cloning. A Laboratory Manual. ColdSpring Harbour Laboratory, 1982).

The reduced stringency conditions used were: prehybridization andhybridization in 30% formamide, 5*SSC, 5*Denhardt, 50 mM phosphatebuffer pH 6.8, 5 mM EDTA, 0.2% SDS and 100:g/ml salmon sperm DNA at42EC. Washings were 4*30 min in 2*SSC, 0.2% SDS at 42EC (Maniatis etal., Molecular Cloning. A Laboratory Manual. Cold Spring HarbourLaboratory, 1982).

Positive 8 phages were purified by replating and hybridization, the cDNAinserts contained in the Bluescript vector present in the 8 ZAPII armswere excised using helper phages obtained from Stratagene (La Jolla,Calif.). The inserts were partially sequenced. One clone designated3(20) showed high homology to the rat GLP-1 receptor and was sequenced(Tabor and Richardson, Proc.Natl.Acad.Sci., USA 84 (1987), 4767-4771) inits entire length. The DNA sequence is shown as SEQ ID No. 3.

From homology analysis (FIG. 7), it was concluded that this cDNA encodedthe 3' part of the human GLP-1 receptor.

The deduced amino acid sequence of the human receptor has 92% identityto the rat GLP-1 receptor in the region from amino acid number 170 toamino acid number 463 (numbers refer to the rat sequence).

The isolated human GLP-1 cDNA does not contain the entire open readingframe at the 5' end. However, a full length clone can easily be obtainedby methods well known to persons skilled in the art. Among thealternative methods of choice, the following examples should bementioned: 1) The human islet cDNA library can either be rescreened witha probe from the 5' end of the already cloned sequence. 2) Anchor-PCR orRACE (Rapid Amplification of cDNA Ends) (Kriangkum et al., Nucleic AcidsRes. 20 (1992) 3793-3794; Troutt et al., Proc.Natl.Acad.Sci., USA 89(1992), 9823-9825) methodology can be used to clone the remaining 5'sequences from islet RNA. 3) The remaining 5' part can be isolated fromhuman genomic libraries, and DNA fragments considered to representintrons can be identified based on homology to the cDNA of the ratreceptor and deleted by mutagenesis.

After cloning of the 5' end of the open reading frame, this part of thecDNA can be fused to the remaining 3' part of the human GLP-1 receptorcDNA by the use of PCR or through fusion at appropriate restrictionenzyme recognition sequences identified in both the 5' and the 3' parts.

The cDNA encoding the full length open reading frame can be cloned insuitable mammalian expression vectors and transfected into mammaliancell lines for expression. Examples of such suitable cell lines are theCHO and CHL cells, but other mammalian cells will also express receptorsof this type.

It has recently been demonstrated that insect cells (Vasudevan et al.FEBS Lett. 311 (1992), 7-11) and microorganisms like e.g. yeast (King etal., Science 250 (1990), 121-123) can express G-protein coupledreceptors.

Recently frog skin melanophore cells have been used to express G-proteincoupled receptors (Potenza et al, Analytical Biochem., 206, (1992),315-322) and a functional coupling to adenylate cyclase wasdemonstrated.

Other microorganisms like Aspergillus, Bacillus, E. coli might be ableto express these receptors after appropriate genetic engineering andselection.

It is therefore clear to persons skilled in the art that a number ofdifferent expression systems can be designed that will lcad toexpression of a functional receptor molecule.

As demonstrated in Example 3, the rat as well as the human GLP-1receptor can be used in screening assays for detection of new potentialagonist lead structures.

EXAMPLE 3 High throughput screening assay for GLP-1 receptor agonists.

Screening of microbial extracts for secondary metabolites with potentialGLP-1 agonist activity was carried out using the SPA (ScintillationProximity Assay) technology (U.S. Pat. No. 4,568,649, Hart and Greenwalt(Mol. Immunol., 16 (1979) 265-267), Udenfriend et al(Proc.Natl.Acad.Sci. USA, 82 (1985) 8672-8676). Wheatgerm agglutinin(WGA) coated SPA beads developed by Amersham International were used(U.S. Pat. No. 4,568,649, European patent 0154734, Japanese patent appl.84/52452). The WGA coat allows GLP-1 receptor bearing membranes to beimmobilized on the SPA beads. Membranes used in the screening assay wereprepared from a CHL, (ATTC CCL39) cell line expressing the cloned ratGLP-1 receptor as described in in Example 1. Membranes were preparedessentially as decribed by Unden et al (Eur. J. Biochem. 145 (1984),525-530). The binding of ¹²⁵ I-GLP-1(7-36)amide to such immobilizedreceptors brings the tracer in close proximity to the scintillantpresent within the SPA beads resulting in the emission of light. Anyunbound ligand will not generate a signal. Thus under assay conditions amicrobial extract--containing a component capable of binding to theGLP-1 receptor and thereby displacing the tracer--may be identified byvirtue of a reduction in signal intensity.

A high throughput assay was established using 96 well microtiter plates.The assay was optimized with regard to the amounts of WGA particles,membrane and tracer used. (The 125I-GLP-1(7-36)amide tracer was labelledusing the lactoperoxidase method (Morrison et al., Methods Enzymol. 70(1980), 214-219) followed by purification on reverse phase HPLC). Usinga Packard TopCount™ microplate scintillation counter (Packard InstrumentCompany) these optimized conditions resulted in a B₀ of more than 7000cpm. (Non specific binding determined in the presence of 500 nMunlabelled GLP-1(7-36)amide amounts to less than 1000 cpm. IC₅₀ =0.5-1.0nM GLP-1(7-36) amide).

So far 1250 microbial extracts have been screened using the SPA GLP-1receptor assay. The extracts were tested at a final dilution of 1:400.Under these conditions 15 out of the 1250 extracts resulted in areduction of specific counts to below the chosen cut-off level. These 15hits have been further characterized in a secondary assay. Thissecondary assay was designed to test whether cAMP synthesis in a GLP-1receptor bearing cell line can be induced by components in the extract.$-TC3 cells (Hanahan et al., Nature 315 (1985) 115-122) and Efrat et al(Proc.Natl.Acad.Sci. USA 85 (1988) 9037-9041) grown in 96-wellmicrotiter plates were exposed to extracts diluted in culture media.After 20 min at 37° C. the cells were lysed by addition of acid and thecAMP concentration determined using the cyclic AMP SPA system (AmershamInternational). Of the 15 primary hits tested in this secondary assay, 5extracts have been found to significantly increase the cAMP level in$-TC3 cells.

It has thus been demonstrated that it is feasible that the screeningapproach described in this patent application can result in theisolation of natural compounds with GLP-1 agonist activity. The use ofsuch compunds as lead structures for a medicinal chemistry approach willbe of significant importance in the design of novel GLP-1 agonists.

EXAMPLE 4 Functional screening for GLP-1 antagonists.

Baby hamster kidney (BHK) cells expressing the human pancreatic GLP-1receptor were used. Plasma membranes were prepared (Adelhorst et al,1994, J. Biol. Chem. 269, 6275) by homogenization in buffer (10 mmol/lTris-HCL and 30 mmol/l NaCL pH 7.4, containing, in addition, 1 mmol/ldithiothreitol, 5 mg/l leupeptin (Sigma, St. Louis, Mo., USA), 5 mg/lpepstatin (Sigma, St. Louis, Mo., USA), 100 mg/l bacitracin (Sigma, St.Louis, Mo., USA), and 16 mg/l aprotinin (Novo Nordisk A/S, Bagsvaerd,Denmark)). The homogenate was centrifuged on top of a layer of 41 w/v %sucrose. The white band between the two layers was diluted in buffer andcentrifuged. Plasma membranes were stored at -80° C. until used.

The assay was carried out in 96-well microtiter plates in a total volumeof 150 ml. The buffer used was 50 mmol/l Tris-HCl, pH 7.4 with theaddition of 1 mmol/l EGTA, 1.5 mmol/l MgSO₄, 1.7 mmol/l ATP, 20 mM GTP,2 mmol/l 3-isobutyl-1-methylxanthine (IBMX), 0.01% tween-20 and 0.1%human serum albumin (HSA) (Reinst, Behringwerke AG, Marburg, Germany).10⁻⁹ M GLP-1 and compounds to be screened for antagonist activity werediluted in buffer, added to the membrane preparation and the mixture wasincubated for 2 h at 37° C. The reaction was stopped by the addition of25 ml of 0.05 mol/l HCL. Samples were diluted 10 fold before analysisfor cAMP by a scintillation proximity assay (RPA 538, Amersham, UK). Thefollowing results indicate no antagonists were identified

    ______________________________________                                                      fmol cAMP (10)                                                  ______________________________________                                        10.sup.-9 M GLP-1                                                                             26.2                                                          0               4.55                                                          10.sup.-9 M GLP-1                                                                             24.7                                                          0               4.30                                                          10.sup.-9 M GLP-1                                                                             30.4                                                          0               5.98                                                          10.sup.-9 M GLP-1                                                                             33.0                                                          0               6.73                                                          10.sup.-9 M GLP-1 + S1                                                                        22.5                                                          10.sup.-9 M GLP-1 + S1                                                                        43.1                                                          10.sup.-9 M GLP-1 + S1                                                                        41.7                                                          10.sup.-9 M GLP-1 + S2                                                                        42.3                                                          10.sup.-9 M GLP-1 + S2                                                                        37.2                                                          10.sup.-9 M GLP-1 + S2                                                                        41.5                                                          10.sup.-9 M GLP-1 + S3                                                                        38.2                                                          10.sup.-9 M GLP-1 + S3                                                                        28.4                                                          10.sup.-9 M GLP-1 + S3                                                                        34.6                                                          ______________________________________                                    

EXAMPLE 5 Functional screening for GLP-1 agonists.

The assay was carried out as in example 4 except compounds were addedalone and not together with GLP-1. The following results indicate noagonists were identified.

    ______________________________________                                                    fmol cAMP (10)                                                    ______________________________________                                        10.sup.-13 M GLP-1                                                                          5.00                                                            10.sup.-12 M GLP-1                                                                          6.14                                                            10.sup.-11 M GLP-1                                                                          5.03                                                            10.sup.-10 M GLP-1                                                                          23.9                                                            10.sup.-9 M GLP-1                                                                           42.9                                                            10.sup.-8 M GLP-1                                                                           46.8                                                            10.sup.-7 M GLP-1                                                                           57.3                                                            S1            9.86                                                            S2            6.72                                                            S3            7.87                                                            S4            8.52                                                            S5            7.88                                                            S6            8.48                                                            S7            8.16                                                            S8            5.89                                                            S9            4.35                                                            S10           5.64                                                            ______________________________________                                    

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 9                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 3066 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Rat                                                             (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 17..1408                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       TCCTGAGCGCCCCGCCATGGCCGTCACCCCCAGCCTGCTGCGCCTGGCG49                           MetAlaValThrProSerLeuLeuArgLeuAla                                             1510                                                                          CTCCTGCTGCTCGGGGCGGTGGGCAGGGCCGGCCCCCGCCCCCAGGGT97                            LeuLeuLeuLeuGlyAlaValGlyArgAlaGlyProArgProGlnGly                              152025                                                                        GCCACGGTGTCCCTCTCAGAGACAGTGCAGAAATGGAGAGAGTATCGG145                           AlaThrValSerLeuSerGluThrValGlnLysTrpArgGluTyrArg                              303540                                                                        CACCAGTGCCAACGTTTCCTCACGGAAGCGCCACTCCTGGCCACAGGT193                           HisGlnCysGlnArgPheLeuThrGluAlaProLeuLeuAlaThrGly                              455055                                                                        CTCTTCTGCAACCGAACCTTTGATGACTACGCCTGCTGGCCAGATGGG241                           LeuPheCysAsnArgThrPheAspAspTyrAlaCysTrpProAspGly                              60657075                                                                      CCCCCAGGTTCCTTTGTGAATGTCAGTTGCCCCTGGTACCTGCCGTGG289                           ProProGlySerPheValAsnValSerCysProTrpTyrLeuProTrp                              808590                                                                        GCCAGTAGTGTGCTCCAAGGGCATGTGTACCGGTTCTGCACGGCCGAG337                           AlaSerSerValLeuGlnGlyHisValTyrArgPheCysThrAlaGlu                              95100105                                                                      GGTATCTGGCTGCATAAGGACAACTCCAGCCTGCCCTGGAGGGACCTG385                           GlyIleTrpLeuHisLysAspAsnSerSerLeuProTrpArgAspLeu                              110115120                                                                     TCGGAGTGCGAAGAGTCCAAGCAAGGAGAGAGAAACTCCCCTGAGGAA433                           SerGluCysGluGluSerLysGlnGlyGluArgAsnSerProGluGlu                              125130135                                                                     CAGCTCCTGTCGCTGTACATTATCTACACGGTGGGGTACGCACTTTCT481                           GlnLeuLeuSerLeuTyrIleIleTyrThrValGlyTyrAlaLeuSer                              140145150155                                                                  TTCTCTGCCTTGGTCATCGCTTCAGCCATCCTTGTCAGCTTCAGACAC529                           PheSerAlaLeuValIleAlaSerAlaIleLeuValSerPheArgHis                              160165170                                                                     TTGCACTGCACCAGGAACTACATCCACCTGAACCTGTTTGCGTCCTTC577                           LeuHisCysThrArgAsnTyrIleHisLeuAsnLeuPheAlaSerPhe                              175180185                                                                     ATCCTCCGAGCACTGTCCGTCTTCATCAAAGACGCTGCCCTCAAGTGG625                           IleLeuArgAlaLeuSerValPheIleLysAspAlaAlaLeuLysTrp                              190195200                                                                     ATGTATAGCACGGCTGCGCAACAGCACCAGTGGGATGGGCTCCTCTCG673                           MetTyrSerThrAlaAlaGlnGlnHisGlnTrpAspGlyLeuLeuSer                              205210215                                                                     TATCAGGACTCTCTGGGCTGCCGACTGGTGTTCCTGCTCATGCAATAC721                           TyrGlnAspSerLeuGlyCysArgLeuValPheLeuLeuMetGlnTyr                              220225230235                                                                  TGCGTGGCGGCCAACTACTACTGGTTGCTGGTGGAAGGCGTGTATCTG769                           CysValAlaAlaAsnTyrTyrTrpLeuLeuValGluGlyValTyrLeu                              240245250                                                                     TACACACTGCTGGCCTTCTCGGTGTTCTCGGAGCAGCGCATCTTCAAG817                           TyrThrLeuLeuAlaPheSerValPheSerGluGlnArgIlePheLys                              255260265                                                                     CTGTACCTGAGCATAGGCTGGGGAGTTCCGCTGCTGTTCGTTATCCCC865                           LeuTyrLeuSerIleGlyTrpGlyValProLeuLeuPheValIlePro                              270275280                                                                     TGGGGCATTGTCAAGTATCTCTACGAGGACGAGGGTTGCTGGACCAGG913                           TrpGlyIleValLysTyrLeuTyrGluAspGluGlyCysTrpThrArg                              285290295                                                                     AACTCCAACATGAACTATTGGCTCATCATACGCTTGCCCATTCTCTTT961                           AsnSerAsnMetAsnTyrTrpLeuIleIleArgLeuProIleLeuPhe                              300305310315                                                                  GCAATCGGGGTCAACTTCCTTGTCTTCATCCGGGTCATCTGCATCGTG1009                          AlaIleGlyValAsnPheLeuValPheIleArgValIleCysIleVal                              320325330                                                                     ATAGCCAAGCTGAAGGCTAATCTCATGTGTAAGACCGACATCAAATGC1057                          IleAlaLysLeuLysAlaAsnLeuMetCysLysThrAspIleLysCys                              335340345                                                                     AGACTCGCGAAGTCCACTCTGACGCTCATCCCGCTTCTGGGCACGCAT1105                          ArgLeuAlaLysSerThrLeuThrLeuIleProLeuLeuGlyThrHis                              350355360                                                                     GAAGTCATCTTTGCCTTTGTGATGGACGAGCACGCCCGAGGAACCCTA1153                          GluValIlePheAlaPheValMetAspGluHisAlaArgGlyThrLeu                              365370375                                                                     CGCTTCGTCAAGCTGTTCACAGAGCTCTCCTTCACTTCCTTCCAGGGC1201                          ArgPheValLysLeuPheThrGluLeuSerPheThrSerPheGlnGly                              380385390395                                                                  TTTATGGTGGCTGTCTTGTACTGCTTTGTCAACAATGAGGTCCAGATG1249                          PheMetValAlaValLeuTyrCysPheValAsnAsnGluValGlnMet                              400405410                                                                     GAGTTTCGGAAGAGCTGGGAGCGCTGGAGGCTGGAGCGCTTGAACATC1297                          GluPheArgLysSerTrpGluArgTrpArgLeuGluArgLeuAsnIle                              415420425                                                                     CAGAGGGACAGCAGCATGAAACCCCTCAAGTGTCCCACCAGCAGCGTC1345                          GlnArgAspSerSerMetLysProLeuLysCysProThrSerSerVal                              430435440                                                                     AGCAGTGGGGCCACGGTGGGCAGCAGCGTGTATGCAGCCACCTGCCAA1393                          SerSerGlyAlaThrValGlySerSerValTyrAlaAlaThrCysGln                              445450455                                                                     AATTCCTGCAGCTGAGCCCCAGTGCTGCGCTTCCTGATGGTCCTTGCTGCTG1445                      AsnSerCysSer                                                                  460                                                                           GCTGGGTGGCCATCCCAGGTGGGAGAGACCCTGGGGACAGGGAATATGAGGGATACAGGC1505              ACATGTGTGTGCGTGCCCGCACACCACACACACACACACACACACACACACACACACACA1565              CACACACACACACACGCTTTCCTCCTCAAACCTATCAAACAGGCATCGGCATCGGCAGTG1625              CCTCCTGGGACCACAGACACATGTTCTCCAAGGAGAACAGCCTGCTAATTTAATCTCAGG1685              CGACAGGAAGAGAGGAAGAAACAATTGCTGTTAAGACGAGGAGGACTTCTTCCTGTTAAA1745              GCTGCAAGGCCCTTGGGGTTCCCTCGGACAGAACTGCAAATCAACCCCGGAACTCTCGCT1805              CAAGGGCAATTGCTGACGGGTGGAACTTGGGCTTGCGAGAGGAGGCAGGTCCATGAGAGA1865              CCTGCCCTTGGAACCTCAGCCAGCACAGCGCTCATCAAGGTGAGCTGGCTGTGCTGTGTG1925              CACGGCTGGGGTTGTCACCTACATCAGCCTTCCTCTCGGACAAGAGGCTTTTCTCTGTGC1985              ATCTGGAGTGCCGCCATTCCTCCATCTGCCCGTTCATCCGCCATCCTGTCTTTGCCTTGG2045              GGAGGGGGAGGTTTGTTGAAGTCATGCCGTGCAGCTCTTTCTGGAAATATCTGTGGATGG2105              TGTTGAAGATAAGCATGGGGGAGATACAACAGAGGCAGTCTTTGCCCATGGCCACTTCTT2165              GCCTGGTCCTTTAAGCCACTTTGCTGCTTGGTTTCTGCCCTGCATGGGTACTACTAGGGC2225              AGGTCCCAAGTTGAGAAGCCCAGAGGTGAGGTGTGAACCCTCAGTTCTGTTGTAAAGATG2285              CTCAAATACCCTCTAAGGTTCATCTAAAGGAGTAACCTGCCTAGGGGTGCTGTTGACCTG2345              AAATCAAGAGGACCAAAGGATCCATTGCCAACACCCCCCATCCCCCACACACACCTCATC2405              TGTGACCAGAGTCTATGCTTTGAATCAGAATGGGCTATATCCTCTGACCTCAGAGGCTAT2465              GACCCAGAAGAGATTCTTCCCTGAATCCTCCCACTTTGCACACATATAGACTTTATCCTT2525              CTTCACTCTGTGTCTATTCAAACGTATAATTCTGGTTTCTCTCACCCCACGGAAGAACTA2585              GATCACAGCAACTGTTATGTTTGAGGGAGTGGGGGAGAAGGTGATTGATTTGACCCCCTC2645              TCCCCCACCGGTGTTGATAAGTAGCGTCTGTCCCACCTCCAGACTCCACCCACACATAAT2705              GAGCAGCACATAGACCAGGATGGGGGGGGTGGTATATCATGCTTGCCCTCCTCCAACCAC2765              TATGAGAAGGCTAGCAGAAGACACCACTGCACAGACCCAAGTCCAAGGACTGCCTCCCAG2825              GGAATTAGGCAGTGACTTCCTAGAGGCCAAGAAAGACTCCAAGAGCTGGAGAAGAATCCT2885              AGTCGATCTGGATCTCTTTTGAGGTTGGGGTTGGGGTGGCTTTCAATGGATTCTCTCATG2945              AGGCTTATCTCTCCCTCATCCCGTGGAGAGTGGGGGACCCTCCCTAGTGCTCACACTAGA3005              CACTGTGCCCCTTGGAGAGGCATAAGGCATGTATGGGAGATAATAATGGGCTATAAAACA3065              T3066                                                                         (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 463 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       MetAlaValThrProSerLeuLeuArgLeuAlaLeuLeuLeuLeuGly                              151015                                                                        AlaValGlyArgAlaGlyProArgProGlnGlyAlaThrValSerLeu                              202530                                                                        SerGluThrValGlnLysTrpArgGluTyrArgHisGlnCysGlnArg                              354045                                                                        PheLeuThrGluAlaProLeuLeuAlaThrGlyLeuPheCysAsnArg                              505560                                                                        ThrPheAspAspTyrAlaCysTrpProAspGlyProProGlySerPhe                              65707580                                                                      ValAsnValSerCysProTrpTyrLeuProTrpAlaSerSerValLeu                              859095                                                                        GlnGlyHisValTyrArgPheCysThrAlaGluGlyIleTrpLeuHis                              100105110                                                                     LysAspAsnSerSerLeuProTrpArgAspLeuSerGluCysGluGlu                              115120125                                                                     SerLysGlnGlyGluArgAsnSerProGluGluGlnLeuLeuSerLeu                              130135140                                                                     TyrIleIleTyrThrValGlyTyrAlaLeuSerPheSerAlaLeuVal                              145150155160                                                                  IleAlaSerAlaIleLeuValSerPheArgHisLeuHisCysThrArg                              165170175                                                                     AsnTyrIleHisLeuAsnLeuPheAlaSerPheIleLeuArgAlaLeu                              180185190                                                                     SerValPheIleLysAspAlaAlaLeuLysTrpMetTyrSerThrAla                              195200205                                                                     AlaGlnGlnHisGlnTrpAspGlyLeuLeuSerTyrGlnAspSerLeu                              210215220                                                                     GlyCysArgLeuValPheLeuLeuMetGlnTyrCysValAlaAlaAsn                              225230235240                                                                  TyrTyrTrpLeuLeuValGluGlyValTyrLeuTyrThrLeuLeuAla                              245250255                                                                     PheSerValPheSerGluGlnArgIlePheLysLeuTyrLeuSerIle                              260265270                                                                     GlyTrpGlyValProLeuLeuPheValIleProTrpGlyIleValLys                              275280285                                                                     TyrLeuTyrGluAspGluGlyCysTrpThrArgAsnSerAsnMetAsn                              290295300                                                                     TyrTrpLeuIleIleArgLeuProIleLeuPheAlaIleGlyValAsn                              305310315320                                                                  PheLeuValPheIleArgValIleCysIleValIleAlaLysLeuLys                              325330335                                                                     AlaAsnLeuMetCysLysThrAspIleLysCysArgLeuAlaLysSer                              340345350                                                                     ThrLeuThrLeuIleProLeuLeuGlyThrHisGluValIlePheAla                              355360365                                                                     PheValMetAspGluHisAlaArgGlyThrLeuArgPheValLysLeu                              370375380                                                                     PheThrGluLeuSerPheThrSerPheGlnGlyPheMetValAlaVal                              385390395400                                                                  LeuTyrCysPheValAsnAsnGluValGlnMetGluPheArgLysSer                              405410415                                                                     TrpGluArgTrpArgLeuGluArgLeuAsnIleGlnArgAspSerSer                              420425430                                                                     MetLysProLeuLysCysProThrSerSerValSerSerGlyAlaThr                              435440445                                                                     ValGlySerSerValTyrAlaAlaThrCysGlnAsnSerCysSer                                 450455460                                                                     (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1909 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 3..887                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       TCAGACACCTGTACTGCACCAGGAACTACATCCACCTGAACCTGTTT47                             ArgHisLeuTyrCysThrArgAsnTyrIleHisLeuAsnLeuPhe                                 151015                                                                        GCATCCTTCATCCTGCGAGCATTGTCCGTCTTCATCAAGGACGCAGCC95                            AlaSerPheIleLeuArgAlaLeuSerValPheIleLysAspAlaAla                              202530                                                                        CTGAAGTGGATGTATAGCACAGCCGCCCAGCAGCACCAGTGGGATGGG143                           LeuLysTrpMetTyrSerThrAlaAlaGlnGlnHisGlnTrpAspGly                              354045                                                                        CTCCTCTCCTACCAGGACTCTCTGAGCTGCCGCCTGGTGTTTCTGCTC191                           LeuLeuSerTyrGlnAspSerLeuSerCysArgLeuValPheLeuLeu                              505560                                                                        ATGCAGTACTGTGTGGCGGCCAATTACTACTGGCTCTTGGTGGAGGGC239                           MetGlnTyrCysValAlaAlaAsnTyrTyrTrpLeuLeuValGluGly                              657075                                                                        GTGTACCTGTACACACTGCTGGCCTTCTCGGTGTTCTCTGAGCAATGG287                           ValTyrLeuTyrThrLeuLeuAlaPheSerValPheSerGluGlnTrp                              80859095                                                                      ATCTTCAGGCTCTACGTGAGCATAGGCTGGGGTGTTCCCCTGCTGTTT335                           IlePheArgLeuTyrValSerIleGlyTrpGlyValProLeuLeuPhe                              100105110                                                                     GTTGTCCCCTGGGGCATTGTCAAGATCCTCTATGAGGACGAGGGCTGC383                           ValValProTrpGlyIleValLysIleLeuTyrGluAspGluGlyCys                              115120125                                                                     TGGACCAGGAACTCCAACATGAACTACTGGCTCATTATCCGGCTGCCC431                           TrpThrArgAsnSerAsnMetAsnTyrTrpLeuIleIleArgLeuPro                              130135140                                                                     ATTCTCTTTGCCATTGGGGTGAACTTCCTCATCTTTGTTCGGGTCATC479                           IleLeuPheAlaIleGlyValAsnPheLeuIlePheValArgValIle                              145150155                                                                     TGCATCGTGGTATCCAAACTGAAGGCCAATGTCATGTGCAAGACAGAC527                           CysIleValValSerLysLeuLysAlaAsnValMetCysLysThrAsp                              160165170175                                                                  ATCAAATGCAGACTTGCCAAGTCCACGCTGACACTCATCCCCCTGCTG575                           IleLysCysArgLeuAlaLysSerThrLeuThrLeuIleProLeuLeu                              180185190                                                                     GGGACTCATGAGGTCATCTTTGCCTTTGTGATGGACGAGCACGCCCGG623                           GlyThrHisGluValIlePheAlaPheValMetAspGluHisAlaArg                              195200205                                                                     GGGACCCTGCGCTTCATCAAGCTGTTTACAGAGCTCTCCTTCACCTCC671                           GlyThrLeuArgPheIleLysLeuPheThrGluLeuSerPheThrSer                              210215220                                                                     TTCCAGGGGCTGATGGTGGCCATCTTATACTGCTTTGTCAACAATGAG719                           PheGlnGlyLeuMetValAlaIleLeuTyrCysPheValAsnAsnGlu                              225230235                                                                     GTCCAGCTGGAATTTCGGAAGAGCTGGGAGCGCTGGCGGCTTGAGCAC767                           ValGlnLeuGluPheArgLysSerTrpGluArgTrpArgLeuGluHis                              240245250255                                                                  TTGCACATCCAGAGGGACAGCAGCATGAAGCCCCTCAAGTGTCCCACC815                           LeuHisIleGlnArgAspSerSerMetLysProLeuLysCysProThr                              260265270                                                                     AGCAGCCTGAGCAGTGGAGCCACGGCGGGCAGCAGCATGTACACAGCC863                           SerSerLeuSerSerGlyAlaThrAlaGlySerSerMetTyrThrAla                              275280285                                                                     ACTTGCCAGGCCTCCTGCAGCTGAGACTCCAGCGCCTGCCCTCCCTGGGGT914                        ThrCysGlnAlaSerCysSer                                                         290                                                                           CCTTGCTGCGGCCGGGTGGCAATCCAGGAGAAGCAGCCTCCTAATTTGATCACAGTGGCG974               AGAGGAGAGGAAAAACGATCGCTGTGAAAATGAGGAGGATTGCTTCTTGTGAAACCACAG1034              GCCCTTGGGGTTCCCCCAGACAGAGCCGCAAATCAACCCCAGACTCAAACTCAAGGTCAA1094              CGGCTTATTAGTGAAACTGGGGCTTGCAAGAGGAGGTGGTTCTGAAAGTGGCTCTTCTAA1154              CCTCAGCCAAACACGAGCGGGAGTGACGGGAGCCTCCTCTGCTTGCATCACTTGGGGTCA1214              CCACCCTCCCCTGTCTTCTCTCAAAGGGAAGCTGTTTGTGTGTCTGGGTTGCTTATTTCC1274              CTCATCTTGCCCCCTCATCTCACTGCCCAGTTTCTTTTTGAGGGCTTGTTGGCCACTGCC1334              AGCAGCTGTTTCTGGAAATGGCTGTAGGTGGTGTTGAGAAAGAATGAGCATTGAGACACG1394              GTGCTCGCTTCTCCTCCAGGTATTTGAGTTGTTTTGGTGCCTGCCTCTGCCATGCCCAGA1454              GAATCAGGGCAGGCTTGCCACCGGGGAACCCAGCCCTGGGGTATGAGCTGCCAAGTCTAT1514              TTTAAAGACGCTCAAGAATCCTCTGGGGTTCATCTAGGGACACGTTAGGAATGTCCAGAC1574              TGTGGGTGTAGGTTACCTGCCACTTCCAGGACGCAGAGGGCCAAGAGAGACATTGCCTCC1634              ACCTCTCCTGAATACTTATCTGTGACCACACGCTGTCTCTTGAGATTTGGATACACTCTC1694              TAGCTTTAGGGGACCATGAAGAGACTCTCTTAGGAAACCAATAGTCCCCATCAGCACCAT1754              GGAGGCAGGCTCCCCCTGCCTTTGAAATTCCCCCACTTGGGAGCTGATATACTTCACTCA1814              CTTTTCTTTATTGCTGTGATAGTCTGTGTGCACAATGGGCAATTCTGACTTCTCCCATCT1874              AGTGAAATGAGCGAAATCATGGTTGTAGTGATCTT1909                                       (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 294 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       ArgHisLeuTyrCysThrArgAsnTyrIleHisLeuAsnLeuPheAla                              151015                                                                        SerPheIleLeuArgAlaLeuSerValPheIleLysAspAlaAlaLeu                              202530                                                                        LysTrpMetTyrSerThrAlaAlaGlnGlnHisGlnTrpAspGlyLeu                              354045                                                                        LeuSerTyrGlnAspSerLeuSerCysArgLeuValPheLeuLeuMet                              505560                                                                        GlnTyrCysValAlaAlaAsnTyrTyrTrpLeuLeuValGluGlyVal                              65707580                                                                      TyrLeuTyrThrLeuLeuAlaPheSerValPheSerGluGlnTrpIle                              859095                                                                        PheArgLeuTyrValSerIleGlyTrpGlyValProLeuLeuPheVal                              100105110                                                                     ValProTrpGlyIleValLysIleLeuTyrGluAspGluGlyCysTrp                              115120125                                                                     ThrArgAsnSerAsnMetAsnTyrTrpLeuIleIleArgLeuProIle                              130135140                                                                     LeuPheAlaIleGlyValAsnPheLeuIlePheValArgValIleCys                              145150155160                                                                  IleValValSerLysLeuLysAlaAsnValMetCysLysThrAspIle                              165170175                                                                     LysCysArgLeuAlaLysSerThrLeuThrLeuIleProLeuLeuGly                              180185190                                                                     ThrHisGluValIlePheAlaPheValMetAspGluHisAlaArgGly                              195200205                                                                     ThrLeuArgPheIleLysLeuPheThrGluLeuSerPheThrSerPhe                              210215220                                                                     GlnGlyLeuMetValAlaIleLeuTyrCysPheValAsnAsnGluVal                              225230235240                                                                  GlnLeuGluPheArgLysSerTrpGluArgTrpArgLeuGluHisLeu                              245250255                                                                     HisIleGlnArgAspSerSerMetLysProLeuLysCysProThrSer                              260265270                                                                     SerLeuSerSerGlyAlaThrAlaGlySerSerMetTyrThrAlaThr                              275280285                                                                     CysGlnAlaSerCysSer                                                            290                                                                           (2) INFORMATION FOR SEQ ID NO: 5:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 449 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Rattus norvegicus                                               (B) STRAIN: Sprague-Dawley                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:                                      MetLeuSerThrMetArgProArgLeuSerLeuLeuLeuLeuArgLeu                              151015                                                                        LeuLeuLeuThrLysAlaAlaHisThrValGlyValProProArgLeu                              202530                                                                        CysAspValArgArgValLeuLeuGluGluArgAlaHisCysLeuGln                              354045                                                                        GlnLeuSerLysGluLysLysGlyAlaLeuGlyProGluThrAlaSer                              505560                                                                        GlyCysGluGlyLeuTrpAspAsnMetSerCysTrpProSerSerAla                              65707580                                                                      ProAlaArgThrValGluValGlnCysProLysPheLeuLeuMetLeu                              859095                                                                        SerAsnLysAsnGlySerLeuPheArgAsnCysThrGlnAspGlyTrp                              100105110                                                                     SerGluThrPheProArgProAspLeuAlaCysGlyValAsnIleAsn                              115120125                                                                     AsnSerPheAsnGluArgArgHisAlaTyrLeuLeuLysLeuLysVal                              130135140                                                                     MetTyrThrValGlyTyrSerSerSerLeuAlaMetLeuLeuValAla                              145150155160                                                                  LeuSerIleLeuCysSerPheArgArgLeuHisCysThrArgAsnTyr                              165170175                                                                     IleHisMetHisLeuPheValSerPheIleLeuArgAlaLeuSerAsn                              180185190                                                                     PheIleLysAspAlaValLeuPheSerSerAspAspValThrTyrCys                              195200205                                                                     AspAlaHisLysValGlyCysLysLeuValMetIlePhePheGlnTyr                              210215220                                                                     CysIleMetAlaAsnTyrAlaTrpLeuLeuValGluGlyLeuTyrLeu                              225230235240                                                                  HisThrLeuLeuAlaIleSerPhePheSerGluArgLysTyrLeuGln                              245250255                                                                     AlaPheValLeuLeuGlyTrpGlySerProAlaIlePheValAlaLeu                              260265270                                                                     TrpAlaIleThrArgHisPheLeuGluAsnThrGlyCysTrpAspIle                              275280285                                                                     AsnAlaAsnAlaSerValTrpTrpValIleArgGlyProValIleLeu                              290295300                                                                     SerIleLeuIleAsnPheIlePhePheIleAsnIleLeuArgIleLeu                              305310315320                                                                  MetArgLysLeuArgThrGlnGluThrArgGlySerGluThrAsnHis                              325330335                                                                     TyrLysArgLeuAlaLysSerThrLeuLeuLeuIleProLeuPheGly                              340345350                                                                     IleHisTyrIleValPheAlaPheSerProGluAspAlaMetGluVal                              355360365                                                                     GlnLeuPhePheGluLeuAlaLeuGlySerPheGlnGlyLeuValVal                              370375380                                                                     AlaValLeuTyrCysPheLeuAsnGlyGluValGlnLeuGluValGln                              385390395400                                                                  LysLysTrpArgGlnTrpHisLeuGlnGluPheProLeuArgProVal                              405410415                                                                     AlaPheAsnAsnSerPheSerAsnAlaThrAsnGlyProThrHisSer                              420425430                                                                     ThrLysAlaSerThrGluGlnSerArgSerIleProArgAlaSerIle                              435440445                                                                     Ile                                                                           (2) INFORMATION FOR SEQ ID NO: 6:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 585 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Didelphis virginiana                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:                                      MetGlyAlaProArgIleSerHisSerLeuAlaLeuLeuLeuCysCys                              151015                                                                        SerValLeuSerSerValTyrAlaLeuValAspAlaAspAspValIle                              202530                                                                        ThrLysGluGluGlnIleIleLeuLeuArgAsnAlaGlnAlaGlnCys                              354045                                                                        GluGlnArgLeuLysGluValLeuArgValProGluLeuAlaGluSer                              505560                                                                        AlaLysAspTrpMetSerArgSerAlaLysThrLysLysGluLysPro                              65707580                                                                      AlaGluLysLeuTyrSerGlnAlaGluGluSerArgGluValSerAsp                              859095                                                                        ArgSerArgLeuGlnAspGlyPheCysLeuProGluTrpAspAsnIle                              100105110                                                                     ValCysTrpProAlaGlyValProGlyLysValValAlaValProCys                              115120125                                                                     ProAspTyrIleTyrAspPheAsnHisLysGlyArgAlaTyrArgArg                              130135140                                                                     CysAspSerAsnGlySerTrpGluLeuValProGlyAsnAsnArgThr                              145150155160                                                                  TrpAlaAsnTyrSerGluCysValLysPheLeuThrAsnGluThrArg                              165170175                                                                     GluArgGluValPheAspArgLeuGlyMetIleTyrThrValGlyTyr                              180185190                                                                     SerIleSerLeuGlySerLeuThrValAlaValLeuIleLeuGlyTyr                              195200205                                                                     PheArgArgLeuHisCysThrArgAsnTyrIleHisMetHisLeuPhe                              210215220                                                                     ValSerPheMetLeuArgAlaValSerIlePheIleLysAspAlaVal                              225230235240                                                                  LeuTyrSerGlyValSerThrAspGluIleGluArgIleThrGluGlu                              245250255                                                                     GluLeuArgAlaPheThrGluProProProAlaAspLysAlaGlyPhe                              260265270                                                                     ValGlyCysArgValAlaValThrValPheLeuTyrPheLeuThrThr                              275280285                                                                     AsnTyrTyrTrpIleLeuValGluGlyLeuTyrLeuHisSerLeuIle                              290295300                                                                     PheMetAlaPhePheSerGluLysLysTyrLeuTrpGlyPheThrLeu                              305310315320                                                                  PheGlyTrpGlyLeuProAlaValPheValAlaValTrpValThrVal                              325330335                                                                     ArgAlaThrLeuAlaAsnThrGluCysTrpAspLeuSerSerGlyAsn                              340345350                                                                     LysLysTrpIleIleGlnValProIleLeuAlaAlaIleValValAsn                              355360365                                                                     PheIleLeuPheIleAsnIleIleArgValLeuAlaThrLysLeuArg                              370375380                                                                     GluThrAsnAlaGlyArgCysAspThrArgGlnGlnTyrArgLysLeu                              385390395400                                                                  LeuLysSerThrLeuValLeuMetProLeuPheGlyValHisTyrIle                              405410415                                                                     ValPheMetAlaThrProTyrThrGluValSerGlyIleLeuTrpGln                              420425430                                                                     ValGlnMetHisTyrGluMetLeuPheAsnSerPheGlnGlyPhePhe                              435440445                                                                     ValAlaIleIleTyrCysPheCysAsnGlyGluValGlnAlaGluIle                              450455460                                                                     LysLysSerTrpSerArgTrpThrLeuAlaLeuAspPheLysArgLys                              465470475480                                                                  AlaArgSerGlySerSerThrTyrSerTyrGlyProMetValSerHis                              485490495                                                                     ThrSerValThrAsnValGlyProArgGlyGlyLeuAlaLeuSerLeu                              500505510                                                                     SerProArgLeuAlaProGlyAlaGlyAlaSerAlaAsnGlyHisHis                              515520525                                                                     GlnLeuProGlyTyrValLysHisGlySerIleSerGluAsnSerLeu                              530535540                                                                     ProSerSerGlyProGluProGlyThrLysAspAspGlyTyrLeuAsn                              545550555560                                                                  GlySerGlyLeuTyrGluProMetValGlyGluGlnProProProLeu                              565570575                                                                     LeuGluGluGluArgGluThrValMet                                                   580585                                                                        (2) INFORMATION FOR SEQ ID NO: 7:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 482 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Sus scrofa                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:                                      MetArgPheThrLeuThrArgTrpCysLeuThrLeuPheIlePheLeu                              151015                                                                        AsnArgProLeuProValLeuProAspSerAlaAspGlyAlaHisThr                              202530                                                                        ProThrLeuGluProGluProPheLeuTyrIleLeuGlyLysGlnArg                              354045                                                                        MetLeuGluAlaGlnHisArgCysTyrAspArgMetGlnLysLeuPro                              505560                                                                        ProTyrGlnGlyGluGlyLeuTyrCysAsnArgThrTrpAspGlyTrp                              65707580                                                                      SerCysTrpAspAspThrProAlaGlyValLeuAlaGluGlnTyrCys                              859095                                                                        ProAspTyrPheProAspPheAspAlaAlaGluLysValThrLysTyr                              100105110                                                                     CysGlyGluAspGlyAspTrpTyrArgHisProGluSerAsnIleSer                              115120125                                                                     TrpSerAsnTyrThrMetCysAsnAlaPheThrProAspLysLeuGln                              130135140                                                                     AsnAlaTyrIleLeuTyrTyrLeuAlaIleValGlyHisSerLeuSer                              145150155160                                                                  IleLeuThrLeuLeuIleSerLeuGlyIlePheMetPheLeuArgSer                              165170175                                                                     IleSerCysGlnArgValThrLeuHisLysAsnMetPheLeuThrTyr                              180185190                                                                     ValLeuAsnSerIleIleIleIleValHisLeuValValIleValPro                              195200205                                                                     AsnGlyGluLeuValLysArgAspProProIleCysLysValLeuHis                              210215220                                                                     PhePheHisGlnTyrMetMetSerCysAsnTyrPheTrpMetLeuCys                              225230235240                                                                  GluGlyValTyrLeuHisThrLeuIleValValSerValPheAlaGlu                              245250255                                                                     GlyGlnArgLeuTrpTrpTyrHisValLeuGlyTrpGlyPheProLeu                              260265270                                                                     IleProThrThrAlaHisAlaIleThrArgAlaValLeuPheAsnAsp                              275280285                                                                     AsnCysTrpLeuSerValAspThrAsnLeuLeuTyrIleIleHisGly                              290295300                                                                     ProValMetAlaAlaLeuValValAsnPhePhePheLeuLeuAsnIle                              305310315320                                                                  LeuArgValLeuValLysLysLeuLysGluSerGlnGluAlaGluSer                              325330335                                                                     HisMetTyrLeuLysAlaValArgAlaThrLeuIleLeuValProLeu                              340345350                                                                     LeuGlyValGlnPheValValLeuProTrpArgProSerThrProLeu                              355360365                                                                     LeuGlyLysIleTyrAspTyrValValHisSerLeuIleHisPheGln                              370375380                                                                     GlyPhePheValAlaIleIleTyrCysPheCysAsnHisGluValGln                              385390395400                                                                  GlyAlaLeuLysArgGlnTrpAsnGlnTyrGlnAlaGlnArgTrpAla                              405410415                                                                     GlyArgArgSerThrArgAlaAlaAsnAlaAlaAlaAlaThrAlaAla                              420425430                                                                     AlaAlaAlaAlaLeuAlaGluThrValGluIleProValTyrIleCys                              435440445                                                                     HisGlnGluProArgGluGluProAlaGlyGluGluProValValGlu                              450455460                                                                     ValGluGlyValGluValIleAlaMetGluValLeuGluGlnGluThr                              465470475480                                                                  SerAla                                                                        (2) INFORMATION FOR SEQ ID NO: 8:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:                                      ArgLeuAlaLys                                                                  (2) INFORMATION FOR SEQ ID NO: 9:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:                                      LysAlaLeuLys                                                                  1                                                                             __________________________________________________________________________

What is claimed is:
 1. A method for detecting the presence of agucagon-like peptide-1 (GLP-1) antagonist, comprising the steps of:(a)exposing a compound in the presence of GLP-1 agonist to a GLP-1 receptorproduced in a heterologous exression system and coupled to a responsepathway under conditions md for a time sufficient to allow binding ofthe compound to the receptor and an associated response through thepathway, wherein the GLP-1 receptor is a GLP-1 tecetor polypeptidehaving the amino acid sequence of a naturally occurring mammalian GLP-1receptor encoded by cDNA molecule isolated from a mammalian library andcomprisig a nucleotide sequence selected from the group consisting of(i) SEQ ID NO:1, (ii) SEQ ID NO:3, and (iii) the sequence of cDNAmolecule capable of specifically hybridizing with a probe having thesequence of the complement of SEQ ID NO: 1 or SEQ ID NO: 3 underconditions of reduced stringency, wherein a probe having the sequence ofthe complement of SEQ ID NO: 1 will specifically identify a cDNAcomprising the sequence shown in SEQ ID NO: 3 in a human cDNA library,said receptor polypeptide capable of binding GLP-1 with K_(D) of lessthan 100 nM; and (b) detecting a reduction in the stimulation of theresponse pathway resulting from the binding of the compound to the GTP-1receptor, relative to the stimulation of the response pathway by theGLP-1 agonist alone and therefrom determiring the presence of a GLP-1antagonist.
 2. The method of claim 1 wherein the response pathway is amembrane-bound adenylate cyclase response pathway.
 3. The method ofclaim 2 wherein the step of detecting comprises measuring a reduction incyclic AMP production by the membrane-bound adenylate cyclase responsepathway.
 4. The method of claim 1 wherein the response pathway includesa luciferase reporter system.
 5. The method of claim 1, wherein theGLP-1 receptor polypeptide has the sequence of a GLP-1 receptor of rator human origin.
 6. The method of claim 1, wherein the expression systemcomprises heterologous DNA comprising the nucleotide sequence shown inSEQ ID NO:
 1. 7. A method for detecting the presence of a GLP-1 agonist,comprising the steps of:(a) exposing a compound to a GLP-1 receptorproduced in a heterologous expression system and coupled to a responsepathway under conditions and for a time sufficient to allow binding ofthe compound to the receptor and an associated response through thepathway, wherein the GLP-1 recptor is a GLP-1 receptor polypeptidehaving the amino acid sequence of a naturally occurring mammalian GLP-1receptor encoded by cDNA molecule isolated from a mammalian library andcomprising a nucleotide sequence selected from the group consisting of(i) SEQ ID NO:1, (ii) SEQ ID NO:3, and (iii) the sequence of cDNAmolecule capable of specifically hybridizing with a probe having thesequence of the complement of SEQ NO: 1 or SEQ ID NO: 3 under conditionsof reduced stringency, wherein a probe having the sequence of thecomplement of SEQ ID NO: 1 will specifically identify a cDNA comprisingthe sequence shown in SEQ ID NO: 3 in a human cDNA library, saidreceptor polypeptide capable of binding GLP-1 with K_(D) of less than100 nM; and (b) detecting stimulation of the response pathway relativeto the non stimulated pathway.
 8. The method of claim 7 wherein theresponse pathway is a membrane-bound adenylate cyclase response pathway.9. The method of claim 8 wherein the step of detecting comprisesmeasuring a production in cyclic AMP production by the membrane.
 10. Themethod of claim 7 wherein the response pathway includes a luciferasereporter system.
 11. The metlod of claim 7, wherein the GLP-1 receptorpolypeptide has the sequence of a GLP-1 receptor of rat or human origin.12. The method of claim 7, wherein the expression system comprisesheterologous DNA comprising the nucleotide sequence shown in SEQ ID NO:1.